Interaction of Staphylococcus aureus and Host Cells upon Infection of Bronchial Epithelium during Different Stages of Regeneration

The primary barrier that protects our lungs against infection by pathogens is a tightly sealed layer of epithelial cells. When the integrity of this barrier is disrupted as a consequence of chronic pulmonary diseases or viral insults, bacterial pathogens will gain access to underlying tissues. A major pathogen that can take advantage of such conditions is Staphylococcus aureus, thereby causing severe pneumonia. In this study, we investigated how S. aureus responds to different conditions of the human epithelium, especially nonpolarization and fibrogenesis during regeneration using an in vitro infection model. The infective process was monitored by quantification of the epithelial cell and bacterial populations, fluorescence microscopy, and mass spectrometry. The results uncover differences in bacterial internalization and population dynamics that correlate with the outcome of infection. Protein profiling reveals that, irrespective of the polarization state of the epithelial cells, the invading bacteria mount similar responses to adapt to the intracellular milieu. Remarkably, a bacterial adaptation that was associated with the regeneration state of the epithelial cells concerned the early upregulation of proteins controlled by the redox-responsive regulator Rex when bacteria were confronted with a polarized cell layer. This is indicative of the modulation of the bacterial cytoplasmic redox state to maintain homeostasis early during infection even before internalization. Our present observations provide a deeper insight into how S. aureus can take advantage of a breached epithelial barrier and show that infected epithelial cells have limited ability to respond adequately to staphylococcal insults

A global Staphylococcus aureus proteome resource applied to the in vivo characterization of host-pathogen interactions.

Data-independent acquisition mass spectrometry promises higher performance in terms of quantification and reproducibility compared to data-dependent acquisition mass spectrometry methods. To enable high-accuracy quantification of Staphylococcus aureus proteins, we have developed a global ion library for data-independent acquisition approaches employing high-resolution time of flight or Orbitrap instruments for this human pathogen. We applied this ion library resource to investigate the time-resolved adaptation of S. aureus to the intracellular niche in human bronchial epithelial cells and in a murine pneumonia model. In epithelial cells, abundance changes for more than 400 S. aureus proteins were quantified, revealing, e.g., the precise temporal regulation of the SigB-dependent stress response and differential regulation of translation, fermentation, and amino acid biosynthesis. Using an in vivo murine pneumonia model, our data-independent acquisition quantification analysis revealed for the first time the in vivo proteome adaptation of S. aureus. From approximately 2.15 × 1

Adaptive immune response to lipoproteins of Staphylococcus aureus in healthy subjects

Staphylococcus aureus is a frequent commensal but also a dangerous pathogen, causing many forms of infection ranging from mild to life-threatening conditions. Among its virulence factors are lipoproteins, which are anchored in the bacterial cell membrane. Lipoproteins perform various functions in colonization, immune evasion, and immunomodulation. These proteins are potent activators of innate immune receptors termed Toll-like receptors 2 and 6. This study addressed the specific B-cell and T-cell responses directed to lipoproteins in human S. aureus carriers and non-carriers. 2D immune proteomics and ELISA approaches revealed that titers of antibodies (IgG) binding to S. aureus lipoproteins were very low. Proliferation assays and cytokine profiling data showed only subtle responses of T cells; some lipoproteins did not elicit proliferation. Hence, the robust activation of the innate immune system by S. aureus lipoproteins does not translate into a strong adaptive immune response. Reasons for this may include inaccessibility of lipoproteins for B cells as well as ineffective processing and presentation of the antigens to T cells

Comparative analysis of Salivette® and paraffin gum preparations for establishment of a metaproteomics analysis pipeline for stimulated human saliva

The value of saliva as a diagnostic tool can be increased by taxonomic and functional analyses of the microbiota as recently demonstrated. In this proof-of-principle study, we compare two collection methods (Salivette® (SV) and paraffin gum (PG)) for stimulated saliva from five healthy participants and present a workflow including PG preparation which is suitable for metaproteomics.Peer Reviewe

Metaproteomics analysis of microbial diversity of human saliva and tongue dorsum in young healthy individuals

Background: The human oral microbiome influences initiation or progression of diseases like caries or periodontitis. Metaproteomics approaches enable the simultaneous investigation of microbial and host proteins and their interactions to improve understanding of oral diseases. Objective: In this study, we provide a detailed metaproteomics perspective of the composition of salivary and tongue microbial communities of young healthy subjects. Design: Stimulated saliva and tongue samples were collected from 24 healthy volunteers, subjected to shotgun nLC-MS/MS and analyzed by the Trans-Proteomic Pipeline and the Prophane tool. Results: 3,969 bacterial and 1,857 human proteins could be identified from saliva and tongue, respectively. In total, 1,971 bacterial metaproteins and 1,154 human proteins were shared in both sample types. Twice the amount of bacterial metaproteins were uniquely identified for the tongue dorsum compared to saliva. Overall, 107 bacterial genera of seven phyla formed the microbiome. Comparative analysis identified significant functional differences between the microbial biofilm on the tongue and the microbiome of saliva. Conclusion: Even if the microbial communities of saliva and tongue dorsum showed a strong similarity based on identified protein functions and deduced bacterial composition, certain specific characteristics were observed. Both microbiomes exhibit a great diversity with seven genera being most abundant.Peer Reviewe

Impact of different oral treatments on the composition of the supragingival plaque microbiome

Background Antiseptics are used to inhibit oral biofilm growth. However, they affect not only pathogenic but also commensal bacteria, which are a natural barrier against oral diseases. Objective Using a metaproteome approach combined with a standard plaque-regrowth study, this pilot study examined the impact of different concentrations of lactoperoxidase (LPO)-system containing lozenges on early plaque formation, and active biological processes. Design Sixteen orally healthy subjects received four local treatments as a randomized single-blind study based on a cross-over design. Two lozenges containing components of the LPO-system in different concentrations were compared to a placebo and Listerine®. The newly formed dental plaque was analyzed by mass spectrometry (nLC-MS/MS). Results On average 1,916 metaproteins per sample were identified, which could be assigned to 116 genera and 1,316 protein functions. Listerine® reduced the number of metaprotein groups and their relative abundance, confirming the plaque inhibiting effect. The LPO-lozenges triggered mainly higher metaprotein abundances of early and secondary colonizers as well as bacteria associated with dental health but also periodontitis. Functional information indicated plaque biofilm growth. Conclusion The effects of Listerine® and LPO-system containing lozenges used for plaque inhibition are different. In contrast to Listerine®, the lozenges allowed maintenance of a higher bacterial diversity

Proteome data of whole saliva which are associated with development of oral mucositis in head and neck cancer patients undergoing radiotherapy

Saliva as major human body fluid may act as an indicator of oral disease status. Oral mucositis is a common and often treatment-limiting side effect of radiotherapy for head and neck cancer patients. In this dataset, we provide the complete proteome dataset (raw and search files) of the patients at baseline of radiotherapy treatment in patients undergoing radiotherapy analyzed by nano liquid chromatography coupled to mass spectrometry (LC–MS/MS). In the data set, 5323 tryptic peptides were identified which can be assigned to 487 distinct proteins (≥2 peptides). The MS data have been deposited to the ProteomeXchange (“ProteomeXchange provides globally coordinated proteomics data submission and dissemination” [1]) via the PRIDE partner repository with the dataset identifier PRIDE: PXD003230. The data are associated with the previously published work, “Differences in the whole saliva baseline proteome profile associated with development of oral mucositis in head and neck cancer patients undergoing radiotherapy” [2]. Keywords: Prospective study, Oral mucositis, Radiotherapy, Radiochemotherapy, Whole saliva, Head and neck cance

Advanced tool for characterization of microbial cultures by combining cytomics and proteomics

Flow cytometry approaches are applicable to recover sub-populations of microbial cultures in a purified form. To examine the characteristics of each sorted cell population, Omics technologies can be used for comprehensively monitoring cellular physiology, adaptation reactions, and regulated processes. In this study, we combined flow cytometry and gel-free proteomic analysis to investigate an artificial mixed bacterial culture consisting of Escherichia coli K-12 and Pseudomonas putida KT2440. Therefore, a filter-based device technique and an on-membrane digestion protocol were combined in conjunction with liquid chromatography and mass spectrometry. This combination enabled us to identify 903 proteins from sorted E. coli K-12 and 867 proteins from sorted P. putida KT2440 bacteria from only 5×10 cells of each. Comparative proteomic analysis of sorted and non-sorted samples was done to prove that sorting did not significantly influence the bacterial proteome profile. We further investigated the physicochemical properties, namely M , pI, hydropathicity, and transmembrane helices of the proteins covered. The on-membrane digestion protocol applied did not require conventional detergents or urea, but exhibited similar recovery of all protein classes as established protocols with non-sorted bacterial samples

Low Dose Proteasome Inhibition Affects Alternative Splicing

Protein degradation by the ubiquitin proteasome system ensures controlled degradation of structural proteins, signaling mediators, and transcription factors. Inhibition of proteasome function by specific proteasome inhibitors results in dose-dependent cellular effects ranging from induction of apoptosis to protective stress responses. The present study seeks to identify nuclear regulators mediating the protective stress response to low dose proteasome inhibition. Primary human endothelial cells were treated with low doses of the proteasome inhibitor MG132 for 2 h, and proteomic analysis of nuclear extracts was performed. Using a 2-D differential in gel electrophoresis (DIGE) approach, we identified more than 24 splice factors to be differentially regulated by low dose proteasome inhibition. In particular, several isoforms of hnRNPA1 were shown to be increased, pointing toward altered posttranslational modification of hnRNPA1 upon proteasome inhibition. Elevated levels of splice factors were associated with a different alternative splicing pattern in response to proteasome inhibition as determined by Affymetrix exon array profiling. Of note, we observed alternative RNA processing for stress associated genes such as caspases and heat shock proteins. Our study provides first evidence that low dose proteasome inhibition affects posttranscriptional regulation of splice factors and early alternative splicing events

Low Dose Proteasome Inhibition Affects Alternative Splicing

Protein degradation by the ubiquitin proteasome system ensures controlled degradation of structural proteins, signaling mediators, and transcription factors. Inhibition of proteasome function by specific proteasome inhibitors results in dose-dependent cellular effects ranging from induction of apoptosis to protective stress responses. The present study seeks to identify nuclear regulators mediating the protective stress response to low dose proteasome inhibition. Primary human endothelial cells were treated with low doses of the proteasome inhibitor MG132 for 2 h, and proteomic analysis of nuclear extracts was performed. Using a 2-D differential in gel electrophoresis (DIGE) approach, we identified more than 24 splice factors to be differentially regulated by low dose proteasome inhibition. In particular, several isoforms of hnRNPA1 were shown to be increased, pointing toward altered posttranslational modification of hnRNPA1 upon proteasome inhibition. Elevated levels of splice factors were associated with a different alternative splicing pattern in response to proteasome inhibition as determined by Affymetrix exon array profiling. Of note, we observed alternative RNA processing for stress associated genes such as caspases and heat shock proteins. Our study provides first evidence that low dose proteasome inhibition affects posttranscriptional regulation of splice factors and early alternative splicing events